DNA cycle sequencing

ABSTRACT

Method for determining the nucleotide base sequence of a DNA molecule by amplifying that DNA, contacting the products of the amplifying reaction with an alkaline phosphatase, and determining the nucleotide base sequence of any polynucleic acid products of the amplifying reaction.

This application is a continuation of application Ser. No. 07/938,335,filed Aug. 28, 1992, now abandoned which a continuation-in-part ofapplication Ser. No. 07/767,137 filed Sep. 27, 1991.

BACKGROUND OF THE INVENTION

This invention relates to methods for DNA sequencing in which a labelednucleotide is incorporated into an extending DNA molecule during a chaintermination sequencing method.

The sequence of nucleotide bases in a DNA molecule can be determined ina variety of ways. The chain termination method generally involvessynthesizing DNA, complementary to the template strand to be sequenced,by extending a primer able to hybridize to a portion of that templatestrand, with a DNA polymerase. During the synthesis reaction,deoxynucleoside triphosphates (dNTPs) are incorporated to form a DNAfragment until a chain terminating agent, for example, adideoxynucleotide triphosphate (ddNTP) is incorporated. Incorporation ofa ddNTP prevents further DNA synthesis (a process called chaintermination). The size of each DNA fragment synthesized in thisprocedure is then determined by gel electrophoresis, and thisinformation used to determine the sequence of nucleotides in theoriginal template DNA. For example, Tabor and Richardson, U.S. Pat. No.4,795,699, describe a two step sequencing method in which an unlabeledprimer is labeled in a labeling step, and then extended in the presenceof excess dNTPs and a ddNTP in a chain termination step. In the labelingstep a low concentration of dNTPs is provided (one being labeled) toallow a small amount of primer extension.

In the dideoxy sequencing method, the primer may be labeled, forexample, with ³² P, by a process using a polynucleotide kinase. Suchlabeling allows detection of extended primers after gel electrophoresisby autoradiography of the resulting gel. Alternatively, a labeled dNTPmay be incorporated during the process of DNA synthesis, and thepresence of such labeled dNTPs detected by autoradiography or othermeans. To this end, the dNTP may be labeled either radioactively with ³²P or ³⁵ S. In another procedure, the primer can be labeled with one ormore fluorescent moieties for detection by fluorescence. In yet anotherprocedure, the ddNTP may be labeled, for example, with a fluorescentmarker.

Examples of these procedures are provided in the following publications,none of which are admitted to be prior art to the present application:

Murray, 17 Nucleic Acid Research 8889, 1989 describes use of a ³²P-labeled primer. The primer was labeled by use of a polynucleotidekinase and gamma-³² P ATP.

Higuchi and Ochman, 17 Nucleic Acid Research 5865, 1989, describe use ofa kinase-labeled primer for sequencing DNA fragments produced by apolymerase chain reaction (PCR). PCR is a method for amplifying adesired nucleic acid by use of two primers which are extended by a DNApolymerase to produce a pair of DNA fragments complementary to thedesired nucleic acid. These fragments then hybridize to provide thedesired DNA. This process is repeated 15-30 times. If a thermostable DNApolymerase is used, the process is best cycled by maintaining thetemperature of the reaction alternatively at a temperature suitable forprimer extension (70°-75° C.), then at a temperature suitable fordenaturation (90°-100° C.) to separate the DNA fragments and provide atemplate for the next cycle, and then at a temperature suitable forannealing (42°-55° C.) to bind primers to the template.

Straus and Zagursky, 10 BioTechniques 376, 1991, describe use of α-³² PdATP in a sequencing reaction.

Smith et al., 9 BioTechniques 52, 1990, and Adams-Blakesley, 13 Focus56, 1991, describe sequencing of PCR products using a labeled primer.

Khrishnan et al., 19 Nucleic Acid Research 1153, 1991, describe use of alabeled primer for sequencing lambda DNA. The process of chaintermination is repeated (or cycled) several times to allow production ofa large number of products. This cycling is similar to a PCR since thereaction is allowed to proceed at 42°-55° C., then stopped at 95° C.,and started again at 42°-55° C. (the process is called thermal cyclesequencing). No new reagent need be added once the process is started.The cycling ensures that a large proportion of the labeled primer isextended in the chain termination steps.

Spurgeon and Burke, Abstract 125, Human Genome II, Oct. 22, 1990,describe use of a modified Taq DNA polymerase for thermal cyclesequencing in which an end-labeled primer, or an unlabeled primer isused. Fluorescent labeled ddNTPs and radioactive labeling techniqueswere also used.

Mead et al., Abstract No. 99, Human Genome II, Oct. 22, 1990, describesequencing of lambda DNA in a polymerase chain-based reaction, usingradioisotopes and fluorescent labels.

SUMMARY OF THE INVENTION

This invention features a novel DNA sequencing method in which anunlabeled primer is extended in a labeling reaction performed in acyclical manner. Such a method is advantageous over the prior use of ³²P end-labeled primers (prepared using polynucleotide kinase and γ-³² PATP), since no additional reagents (e.g., polynucleotide kinase, andγ-³² P ATP) are required over the usual sequencing reagents, the processof preparing labeled primer is not required, and, since the apparatusused in the method is identical to that which is used for thermal cyclesequencing, the two methods can be conveniently used together. Thisprocedure allows the use of labels not otherwise usable withpolynucleotide kinase, such as α-³⁵ S dNTPs, fluorescent nucleotides,and nucleotides labeled with other useful moieties, such as biotin.

The critical feature of the present invention is the use of at least onelabeled nucleotide in a cycled labeling step. This is generally followedby a cycled termination step. The repeated cycling of primed DNAsynthesis results in production of many more newly synthesized moleculesof DNA than molecules of template DNA used for sequencing. Thus, whilesequencing using customary amounts of template (0.5-1.0 pmol) ispossible when only a single round of replicative synthesis is performed,sequencing using less template DNA (e.g., 0.05 pmol) without a cyclingstep is difficult. Repeated cycling of the labeling step using limitedamounts of template increases the amount of product to a levelsufficient for normal detection procedures, such as autoradiography withovernight exposure.

In the method, at most three of the four required dNTPs for a sequencingreaction are present in the labeling step, with one or more of thembeing labeled. This effectively labels the primer and limits extensionto a known length (i.e., to a length where the missing dNTP is requiredfor further synthesis). In the method, the reaction is cycled between atemperature suitable for chain extension, and one suitable fordenaturation of the extended primer and template molecules, generally 10to 100 times over a period of one and one-half to six hours. After thislabeling step, the reaction is generally divided into four portions andan appropriate chain terminating mix, including the missing dNTP, issupplied to the reaction mixture and cycled between 10 and 200 times atappropriate temperatures for primer extension and denaturing. Noadditional DNA polymerase is required in this step, and the reaction canbe completed in 1.5 to 16 hours.

The method of this invention allows sequencing of as little as 0.01micrograms (0.005 picomoles) of M13 DNA with an 18 to 36 hour exposurefor radioactively-labeled (³⁵ S) material. With double-strandedplasmids, sequence information can be obtained from as little as 0.03micrograms (0.015 pmol), without prior denaturation of the plasmid DNAwith alkali. In addition, reliable sequence data can be obtaineddirectly from a single M13 plaque, without further growth of the phage,by simply picking the plaque into a reaction vessel and adding theappropriate reagents. In addition, sequences can be obtained from singlecolonies of bacteria containing plasmids having the f1 replicationorigin (e.g., pTZ18R or pUC118) with a helper phage (M13 K07), or evenfor plasmids without helper phage.

Because the invention allows sequencing of small quantities of template,the need to grow clones and purify DNA from them is unnecessary, and thechance of adding impurities into the sequencing reaction along with thetemplate DNA is thus reduced. In addition, because the DNA iseffectively end-labeled by this procedure, there is little systematicvariation in band intensity and no tendency for sequences to fade nearthe bottom of an autoradiogram of a sequencing gel.

Furthermore, the invention allows sequencing of plasmid and otherdouble-stranded DNA templates without prior denaturation using alkali.These denaturation procedures are tedious, requiring an hour or more ofskilled manipulation for denaturation and subsequent precipitation.Thus, the invention allows sequencing of DNA using existing apparatuswith little modification to the procedure necessary to perform theclaimed DNA sequencing reaction.

Thus, in a first aspect, the invention features a method for sequencingDNA which includes the following steps: providing a polynucleotideprimer complementary to a region of the DNA, providing the DNA to besequenced, and contacting that primer and DNA together in the presenceof a DNA polymerase and between 1 and 3 dNTPs, at least one of the dNTPsbeing labeled. The primer and DNA are contacted under conditions whichallow extension of the primer by addition of one or more of the dNTPs tothe 3'-end of the primer to form an extended primer. The primer and DNAare then dissociated, or separated, generally by heating, and thecontacting and separating steps repeated a plurality of times (usually10-200 times). Finally, the extended primer is contacted with the DNA inthe presence of a DNA polymerase (which is generally the same polymeraseas used in the initial labeling step), all four dNTPs and a chainterminating agent.

In preferred embodiments, the DNA polymerase is Taq DNA polymerase(e.g., a form of Taq polymerase lacking a 5'-3' exonuclease activity), alow exonuclease form of T7 DNA polymerase (see Tabor and Richardson,U.S. Pat. No. 4,795,699), Klenow, AMV reverse transcriptase, Bst, Tth orVent DNA polymerase, or exo-free forms of such polymerases; the dNTP islabeled with ³⁵ S or a fluorescent, chemiluminescent or other (e.g., aredetectable as a ligand by indirect enzyme-linked assay, e.g., biotin)label; and the final contacting step in the presence of a chainterminating agent is followed by a separation step in which the extendedprimer is separated from the DNA, and those two steps repeated aplurality of times (e.g., between 30 and 200 times) until approximately0.1-1.0 picomole (preferably 0.5 pmole) of labeled extended primer whichis terminated by a dideoxynucleotide at its 3' end is produced.

In other preferred embodiments, the method is used to sequence:

Purified M13 DNA (single stranded, ss),

Purified Plasmid DNA (Double-stranded, ds),

Purified Lambda DNA (ds),

M13 DNA from a plaque or liquid culture (ss),

Plasmid DNA from a colony or liquid culture (ds),

Plasmid-with-f1-ori, preferably, infected with helper phage, from acolony (ss),

PCR product, e.g., gel purified (ds),

PCR product treated w/ExoI and Alk. Phos. (ds),

Asymmetric PCR product treated with Alk. Phos. (ss),

Purified Cosmid DNA, and

DNA from other sources e.g., yeast or bacterial genomic DNA or otherphage DNAs.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

DRAWINGS

FIG. 1 is a diagrammatic representation of a DNA sequencing procedure ofthis invention;

FIGS. 2-7 are copies of autoradiograms obtained from DNA sequencingexperiments using a variety of conditions described infra, and

FIGS. 8A and 8B are graphs of data from an Applied Biosystems instrumentshowing results of a sequencing experiment.

There follow examples of methods of the invention. These examples arenot limiting in the invention, and those of ordinary skill in the artwill recognize that the components used in the reactions may be readilysubstituted with equivalent reagents known in the art.

METHODS

Referring to FIG. 1, the first step in the method is to examine thesequence of the template (usually vector sequence) and choose acombination of primer and labeling nucleotides. The primer is chosen bystandard criteria, well known in the art, to prime DNA synthesis nearthe sequence of interest. The primer is typically a syntheticoligonucleotide 16-25 nucleotide bases in length.

A mixture of three nucleotides (of the possible four: DATP, dCTP, dGTPand dTTP) is chosen as follows: The first place where dAMP will beincorporated downstream from the 3' terminus of the primer is located.Similarly, the first sites for incorporation of dGMP, dCMP and dTTP arefound. One of these sites will be farther from the primer than all theothers. The nucleotide required at this site is omitted during thelabeling phase of the protocol (dCTP in FIG. 1). One or more of theremaining three nucleotides must be labeled (e.g., α-³² P, α-³⁵ S or afluorescent molecule). A combination is chosen so that at least 2labeled nucleotides will be incorporated prior to termination at thepoint where the missing nucleotide terminates extension. In FIG. 1,there are four dAMP nucleotides incorporated prior to the first sitewhere dCMP would be required. Thus, a labeled dATP is a good choice inthis example.

A suitable combination of primer and nucleotide mix can usually be foundwithout difficulty, however, vectors can be specifically engineered foroptimal labeling of sequences. If necessary the choice of which DNTP toleave out of the labeling mix can be determined empirically by runningtest labelings.

EXAMPLE 1 Sequencing with Purified M13 DNA or Purified Plasmid DNA

The following components were added to a microcentrifuge vial (0.4 ml)which was inserted into a thermocycler machine (e.g., Perkin-Elmer DNAThermal Cycler): 0.005 pmol or more M13 DNA (e.g., M13mp18, 0.01 μg), or0.03 μg double-stranded plasmid DNA (e.g., pUC19); 2 μl ΔTAQ ReactionBuffer (United States Biochemical Corporation, Cleveland, Ohio, 260 mMTris-HCl, pH 9.5 65 mM MgCl₂); 1 μl 3.0 μM dGTP; 1 μl 3.0 μM dTTP; 0.5μl (5 μCi) of α-³⁵ S!dATP (about 1200 Ci/mmol); 1 μl -40 primer (0.5 μM;0.5 pmol/μl 5'GTTTTCCCAGTCACGAC-3'); 2 μl ΔTAQ DNA polymerase 32 U/μl(from United States Biochemical Corporation, Cleveland, Ohio, in 20 mMTris (pH 8.5), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% NP-40, 0.5%TWEEN-20 and 50% glycerol, diluted 8 fold in dilution buffer (10 mMTris-HCl pH 8.0, 1 mM 2-mercaptoethanol, 0.5% TWEEN-20, 0.5% NP-40));and water to a total volume of 17.5 μl.

These components were mixed and overlaid with 10 μl light mineral oil(United States Biochemical Corporation). The vial was placed in thethermocycler and 30-100 cycles (more than 60 cycles is unnecessary) from45° C. for 1 minute to 70° C. for 0.5 minute performed. (Temperaturescan be cycled from 55°-70° C., if desired, or even a constanttemperature of about 62° C. maintained (depending on the meltingtemperature of the primer and template to be sequenced). Such isothermallabeling is possible, but takes as long to complete (about 4-6 hours) ascycled temperatures.) The temperatures may be adjusted if the meltingtemperature of the primer/template is significantly higher or lower, butthese temperatures work well for most primer/templates. A temperature of95° C. can be used instead of 70° C. with equivalent results. This stepcan be completed in about 3 minutes per cycle.

Four vials were labeled A, C, G, and T, and filled with 4 μl of thecorresponding ΔTAQ termination mix: ddA termination mix (15 μM eachdATP, dCTP, dGTP, dTTP, 300 μM ddATP); ddT termination mix (15 μM eachdATP, dCTP, dGTP, dTTP, 450 μM ddTTP); ddC termination mix (15 μM eachdATP, dCTP, dGTP, dTTP, 225 μM ddCTP); ddG termination mix (15 μM eachdATP, dCTP, dGTP, dTTP, 22.5 μM ddGTP) (United States BiochemicalCorporation, Cleveland, Ohio). No additional enzyme is added to thetermination vials. The enzyme carried in from the prior step issufficient.

The cycled labeling reaction was divided equally among the fourtermination vials (4 μl to each termination reaction vial), and overlaidwith 10 μl of light mineral oil.

The four vials were placed in the thermocycler and 30-200 cycles (morethan 60 cycles is unnecessary) performed from 95° C. for 15 seconds, 55°C. for 30 seconds, and 72° C. for 120 seconds. This step wasconveniently completed overnight. Other times and temperatures are alsoeffective.

Six μl of reaction mixture was removed (avoiding oil), 3 μl of StopSolution (95% Formamide 20 mM EDTA, 0.05% Bromophenol Blue, 0.05% XyleneCyanol FF) added, and heated briefly to 70°-80° C. immediately prior toloading on a sequencing gel. Autoradiograms required an 18-36 hourexposure using Kodak XAR-5 film.

Referring to FIG. 2, the indicated amount of purified M13mp18 DNA(0.001-0.1 μg, in lanes E-A respectively) was sequenced using the -40primer and nucleotides as described above. The labeling step was cycled30 times (45° C. to 95° C.) and the termination step was cycled 35 times(95° C., 55° C., and 72° C.). The result in lane F is an example whereall four dNTPs were present during the cycled labeling step (3 pmoleach).

When the above labeling procedure is performed without cycling (usingthe standard protocol for the TAQuence sequencing kit, United StatesBiochemical Corporation), 0.1 μg M13mp18 template DNA yields anautoradiogram where bands representing the first 200 bases from theprimer are absent. The use of less DNA yields even fainter sequence.

EXAMPLE 2 Direct Sequencing of M13 Plaque

An M13 plaque (M13mp18) was picked from an 18-36 hour-incubated plateusing a large opening pipette tip. More phage can be obtained using atip with the tip cut back to enlarge the opening to about 1.5 mmdiameter. A small amount of soft agar adheres to the tip. The soft agarwas mixed well (repeated sucking up and pushing out the liquid in thepipette) with 2 μl of reaction buffer and 10 μl of water. The tube wascapped and heated for 10 minutes at 95°-100° C., and quickly spun tocollect any condensation. 12 μl was then quickly (so the agar does notsolidify) removed for sequencing. The procedure outlined in Example 1was then used for sequencing.

Referring to FIG. 4, the sequence of DNA from a single plaque of M13mp18picked from a lawn of JM101 is shown. The DNA was extracted from theplaque by heating in sequencing buffer at 95°-100° C. for 10 minutes.The DNA was sequenced using the -40 primer and nucleotides as describedabove. The labeling step was cycled 99 times (45° C. to 95° C.) and thetermination step was cycled 198 times (95° C., 55° C., and 72° C.).

EXAMPLE 3 Direct Sequencing of a Colony Containing a Plasmid

A colony containing a cloning vector (e.g., pUC19) is picked from a 36hour-incubated plate (L broth) using a sterile wire or loop, suspendedin 2 μl of reaction buffer and 10 μl of water, and mixed well. Thecolony was heated at 95°-100° C. for 20 minutes, briefly spun tosediment the debris, and 12 μl of liquid removed for sequencing. Theprocedure outlined in Example 1 was then used for sequencing.

Referring to FIG. 3, results of cycled-labeling sequencing of plasmid(pUC19) DNA are shown. The indicated amount of purified pUC19 DNA(0.01-3 μg) was sequenced using the -40 primer and nucleotides asdescribed above. The labeling step was cycled 85 times (45° C. to 95°C.) and the termination step was cycled 198 times (95° C., 55° C. and72° C.).

EXAMPLE 4 Direct Sequencing of a Colony Containing a Plasmid andInfected with Helper Phage

Infection of a cell containing a plasmid with the f1 origin with helperphage results in the production of large amounts of phage-like particlescontaining one strand of the plasmid DNA. (The strand produced isdetermined by the orientation of the f1 origin). These are secreted fromthe cell. Helper phage particles are also produced in lesser quantity(due to an alteration in the helper phage origin) but these do notsequence with the primer used. Thus, when a colony is infected withhelper phage more plasmid DNA (but only one strand) is available forsequencing. Plasmid sequencing from a single colony is marginal,probably because it is difficult to obtain sufficient template DNAroutinely. Infection with a helper phage produces more template, makingsequencing possible.

Host strain JM101 was transformed with a plasmid vector (pTZ18U; Mead etal., 13 Nucleic Acid Research 1103, 1985) which contains the replicationorigin from bacteriophage f1. After incubating the competent cells withthe plasmid at 0° C. (45 minutes) and 42° C. (2 minutes), the cells werediluted in L Broth and incubated at 37° C. for 30 minutes. M13 helperphage M13KO7 (Vierra and Messing, 153 Meth. Enz. 3, 1987; 10 μl of 10¹⁰pfu/ml) was added, and incubation continued at 37° C. for 20 minutes.Transformants were then plated on media containing ampicillin andkanamycin. After incubation for 18-36 hours, a single colony was picked.

A colony from an 18-36 hour-incubated plate was picked using a sterilewire or loop, suspended in 2 μl of reaction buffer and 10 μl of water,and mixed well. The mixture was heated at 95°-100° C. for 20 minutes;alternatively, it was heated at 37° C., for 30 minutes centrifugedbriefly, and the supernatant incubated at 95°-100° C. for 10 minutes.The supernatant was then briefly spun to sediment the debris, and 12 μlremoved for sequencing as above.

Referring to FIG. 5, the sequence of DNA from a single colony of JM101containing pTZ18U infected with M13KO7 is shown. The DNA was extractedfrom the colony by heating in sequencing buffer at 95°-100° C. for 10minutes. The DNA was sequenced using the -40 primer and nucleotides asdescribed above. The labeling step was cycled 99 times (45° C. to 95°C.) and the termination step was cycled 198 times (95° C., 55° C., and72° C.).

EXAMPLE 5 Sequencing of DNA Extracted from Bacteriophage Lambda

Purified DNA from bacteriophage lambda (1 μg in 5 μl H₂ O) was mixedwith 1 μl (0.5 pmol) primer (SEQ. ID. NO. 1:5'-GGTTATCGAAATCAGCCACAGCGCC; corresponding to bases 7630-7606 inbacteriophage lambda), 2 μl ΔTAQ buffer, 1 μl 3 μM dGTP, 1 μl 3 μM dTTP,0.5 μl α-³⁵ S dCTP (1200 Ci/mmol, 10 μCi/μl) and diluted ΔTAQ DNApolymerase (2 μl 4 U/μl) and 5 μl H₂ O for a total volume of 17.5 μl.This mixture was incubated in the DNA Thermal Cycler at 50° C. for 60seconds, then 90° C. for 30 seconds for 99 cycles. Using this mixture ofnucleotides, the primer was extended 7 bases with the sequence (SEQ. ID.NO. 2: TCCCGTT with the C's labeled).

The mixture was then divided into four parts, combining one part with 4μl ddGTP termination mix (15 μM each dATP, dCTP, dGTP, dTTP; 22.5 μMddGTP), a second with 4 μl ddATP termination mix (15 μM each DATP, dCTP,dGTP, dTTP; 300 μM ddATP), the third with 4 μl ddTTP termination mix (15μM each DATP, dCTP, dGTP, dTTP; 450 μM ddTTP) and the fourth with 4 μlddCTP termination mix (15 μM each dATP, dCTP, dGTP, dTTP; 225 μM ddCTP).The mixtures were overlaid with 10 μl mineral oil and the vials capped.These four vials were then placed in the thermal cycler and subjected to198 cycles at 95° C., 15 seconds, 55° C., 30 seconds and 72° C., 120seconds.

Upon completion of the cycles overnight, 6 μl of the aqueous phase wasremoved from each vial, and 3 μl of stop solution (95% Formamide, 20 mMEDTA, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol FF) added. Themixtures were heated to 75° C. for 2 minutes and applied to thesequencing gel. Sequencing required only 1-2 μg (0.03-0.05 pmol).

Referring to FIG. 6, the sequence of DNA from bacteriophage lambda DNAusing a cycled labeling step protocol is shown. 0.5-5 μg of lambda DNAwas used as indicated in the Figure. The autoradiogram shown is theresult of a 60 hour exposure.

EXAMPLE 6 Sequencing DNA Produced by a Polymerase Chain Reaction

Upon completion of a polymerase chain reaction, the reaction mixturestill contains excess primer, dNTPs and non-specifically amplifiedsingle-stranded DNAs. These are typically removed from the desireddouble-stranded product by precipitation, gel purification or otherphysical technique. These procedures are usually time-consuming andrequire manual manipulations for centrifugations or gel electrophoresis.I have discovered that the undesirable single-stranded DNA and primercan be removed by brief incubation with exonuclease I. Similarly, theexcess dNTPs (which would interfere with the labeling portion of thesequencing process) can be removed by simultaneous treatment withalkaline phosphatase. Both enzymes can be inactivated by heatingbriefly. Subsequently, the PCR product can be sequenced using themethods outlined in Example 1.

Specifically, PCR was performed as outlined in the Perkin-Elmer CetusGENEAMP kit using 200 μM dNTPs, 100 pmol each primer, 1 ng template DNAand buffer in a total volume of 100 μl. Template DNA was pT7L-21 (aclone of a modified Tetrahymena ribozyme in pUC18; Zaug et al., 27Biochemistry 8924-8931, 1988) and the primers were -40 universalsequencing primer (SEQ. ID. NO. 3: 5'-GTTTTCCCAGTCACGAC) and reversesequencing primer (SEQ. ID. NO. 4: 5'-TTCACACAGGAAACAG). These primersyield a product 518 nucleotides in length. The polymerase chain reactionwas performed using 30 cycles of 94° C. for 1 minute, 37° C. for 1minute and 72° C. for 2 minutes.

An aliquot (10 μl) of the PCR mixture was removed. Exonuclease I (USBProduct No. 70013; 1 μl of 10 U/μl) and shrimp alkaline phosphatase (USBProduct No. 70092, 1 μl of 1 U/μl) were added. This mixture wasincubated at 37° C. for 15 minutes, followed by heat inactivation at 70°C. for 15 minutes. This mixture was diluted to 200-2000 μl with TEbuffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), and 10 μl used forcycle-sequencing according to the method of example 1 using the -40primer. Control experiments in which the treatments with eitherexonuclease I or shrimp alkaline phosphatase are omitted failed toprovide good sequence data.

Referring to FIG. 7, the DNA sequence of the product of a polymerasechain reaction using a cycled labeling step protocol is shown. Thelabeling step was cycled 65 times (45° C. to 70° C.) and the terminationstep was cycled 198 times (95° C., 55° C. and 72° C.). Aliquots of thePCR reaction mixture were treated with exonuclease I (lanes A and C)and/or shrimp alkaline phosphatase (lanes B, C and D) as describedabove. The enzymes were then heat-inactivated and the DNA diluted20-fold. This was used as template (10 μl) using -40 primer andnucleotides as described above. Pre-treatment with both exonuclease andalkaline phosphatase yields the best sequence (C).

EXAMPLE 7 Cycle Sequencing with Fluorescein-labeled dUTP and FluorescentDetection

In this example, the radioactive label is replaced by non-radioactive,fluorescein-labeled nucleotide (fluorescein-dUTP), Boeringer MannheimBiochemicals). Cycle sequences were run using either single-stranded M13DNA or double-stranded pUC18 plasmid DNA as template. The cycledlabeling step was run using the universal (-40) sequencing primer in thepresence of DATP, dGTP and fluorescein-dUTP. The reaction products wereapplied to a sequencing electrophoresis gel mounted in an ABI model 373ADNA sequencing instrument which detects sequencing products byfluorescence.

For the cycled labeling step, 0.05 pmole (or more) M13 DNA (0.125 μg) or0.36 μg double-stranded plasmid DNA (pUC18), 2 μl ΔTaq Reaction Buffer(260 mM Tris-HCl (pH 9.5), 65 mM MgCl₂), 3 μl Fluorescein-dUTP LabelingMix (10 μM fluorescein-dUTP, 1 μM dGTP, 1 μM dATP), 1 μl "-40" Primer(0.5 μM, 0.5 pmole/μl 5'-GTTTTCCCAGTCACGAC-3'), 2 μl ΔTaq DNA polymerase(8 U/μl) (in 10 mM Tris-HCl pH 8.0, 1 mM 2-mercaptoethanol, 0.5%TWEEN-20, 0.5% NP-40) and water were combined to a total volume of 18 μlin a 0.5 ml microcentrifuge vial, mixed well and overlaid with 15 μllight mineral oil. The vial was placed in the thermocycler (Perkin-ElmerCetus Co.) and subjected to 30-50 cycles of 95° C. for 15 seconds and60° C. for 30 seconds.

This combination of labeling reaction components allowed for theincorporation of a maximum of 3 fluorescein-dUTP molecules per annealedprimer since dCTP was missing from the polymerization mixture asillustrated below:

Before Labeling Step

Primer

. .GTTTTCCCAGTCACGAC

. .CAAAAGGGTCAGTGCTGCAACATTTTGCTG. . .

Template

After Labeling Step

Primer

. .GTTTTCCCAGTCACGACGUUGUAAAA (U=fluorescein dUMP)

. .CAAAAGGGTCAGTGCTGCAACATTTTGCTG. . .

Template

Following the labeling cycles, four vials were marked A, C, G, and T,and filled with 4 μl of the corresponding ΔTaq termination mix. Then thecycled labeling reaction was divided equally among the four terminationvials (3.5 μl to each termination reaction vial), and overlaid with 15μl of light mineral oil.

The four vials were placed in the thermocycler and 30-50 cyclesperformed from 95° C. for 30 seconds, 60° C. for 60 seconds, and 72° C.for 60 seconds. The reactions were stopped by adding 4 μl of StopSolution (95% Formamide, 20 mM EDTA) to the termination reactionsalready under mineral oil. These samples were heated briefly at 75°-80°C. immediately prior to loading on a sequencing gel. Samples (5 μl) wereloaded in four adjacent lanes (corresponding to one of the fourtermination reactions ddA, ddC, dG, and ddT) on a 6% acrylamide, 7 Murea sequencing gel. Electrophoresis of sequencing reaction products wasconducted using the Applied Biosystems 373A DNA Sequencing instrumentovernight for 16 hours at 35 watts constant power. A high sensitivityfluorescence detector positioned near the bottom of the gel detects thefluorescent bands of DNA as they pass by during electrophoresis. Thefluorescence intensity from each lane was collected using theaccompanying ABI computer software using the filter channel optimal forfluorescein detection. This software was also used to normalize thedata, subtracting baseline drift.

FIG. 8 shows the results of sequencing M13mp18 DNA, displaying theresults covering the region from 175-217 bases from the 5' end of theprimer. In panel A, the raw data from four adjacent lanes is displayed.These are colored as follows: Black, ddG reaction; Blue, ddC reaction;Green, ddA reaction; and Red, ddT reaction. The lower panel B shows thesame results after software analysis which removes background drift andnormalizes the average peak heights. The known sequence of the templateDNA in this region is displayed above the peaks. Manual comparison ofthe graph and the sequence indicates that the method correctly displayedthe sequence of all but one or two of the bases in this region.

EXAMPLE 8 Sequencing Asymmetric PCR Products by Pre-treatment withAlkaline Phosphatase

Normally, the polymerase chain reaction (PCR) is performed with equalconcentrations of two primers, leading to the geometric amplification ofthe sequence between their annealing sites. In principle, each cycle candouble the amount of product DNA in the reaction mixture. The productsof such "symmetric" PCR reactions are linear, double-stranded DNAmolecules. Sometimes primers are added to the amplification reaction atunequal concentrations, with a concentration ratio of 10:1 to 500:1.Such "asymmetric" PCR reactions perform normally during the firstseveral cycles, generating a geometrically increasing amount ofdouble-stranded product DNA. Eventually, the supply of thelower-concentration primer is exhausted. From this point, furtheramplification cycles have only a single primer available, so theyproduce only one strand of product DNA. The concentration of thissingle-stranded product increases linearly with additional cycles. Thusthe reaction products consist of a relatively small amount ofdouble-stranded linear DNA and a larger concentration of single-strandedlinear DNA. This single-stranded DNA can be used as template for DNAsequencing.

Gyllensten and Erlich, 85 Proc. Natl. Acad. Sci. USA 7652 (1988),describe a procedure for sequencing the single-stranded products ofasymmetric PCR. The procedure requires the use of an ultrafilter in acentrifuge followed by drying of the concentrated DNA. This proceduretakes several hours to complete and the use of the filter makes itexpensive. Others (Brow, p. 189 in "PCR Protocols: A Guide to Methodsand Applications", Academic Press, 1990) have described the use ofalcohol precipitation to purify the DNA prior to sequencing. Again, thisprocedure takes several hours to complete. They suggest an alternativewhich requires no DNA purification but which requires the use of labeledprimer and reduced concentrations of dNTPs in the PCR process. Labelingthe primer is an additional, time-consuming and expensive step,especially if only a few sequences are being performed.

The purification, or other steps, is required because the dNTPs used forthe PCR interfere with the sequencing process. Applicant has discoveredthat these dNTPs can be removed much more rapidly, simply andinexpensively by the addition of a small amount of heat-labile alkalinephosphatase. Preferred alkaline phosphatases include the Shrimp AlkalinePhosphatase (United States Biochemical Corporation) and Calf-Intestinealkaline phosphatase which are sensitive to heating of 60°-70° C.

The PCR was performed in 100 μl volume using 50 pmol of one primer and 1pmol of the second primer (50:1 primer ratio) in the buffer supplied inthe GeneAmp kit (Perkin Elmer Cetus Corp.) with 2.5 units of AmpliTaqTaq DNA polymerase. The dNTPs were added to a concentration of 0.2 mM.Template was supplied by a single M13 plaque which contained an insertof approximately 1 Kb in size. The phage was eluted from the plaque in100 μl of 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 5 μl used in the PCR. Thevial was placed in the thermocycler and subjected to 50 cycles of 95°C., 1 minute; 55° C., 1 minute; and 72° C., 2 minutes.

Following amplification, an aliquot (10 μl) was removed and 2 units ofalkaline phosphatase were added. This mixture was incubated at 37° C.for 10 minutes and then the phosphatase inactivated by incubation at 70°C. for 10 minutes.

Following this treatment, an aliquot of 7 μl was removed and sequencedusing the normal procedures for the Sequenase Version 2.0 DNA sequencingkit (United States Biochemical Corporation). Sequences were of excellentquality with the alkaline phosphatase pre-treatment but unusable withoutalkaline phosphatase. This method is completed quickly, requiring onlysimple pipetting steps which can be readily performed by automatedrobots. The reagents are readily available and inexpensive. Similar,high-quality sequences have been obtained using amplified human genomicDNA with several primers.

Other embodiments are within the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GGTTATCGAAATCAGCCACAGCGCC25                                                   (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      TCCCGTT7                                                                      (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      GTTTTCCCAGTCACGAC17                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      TTCACACAGGAAACAG16                                                            __________________________________________________________________________

I claim:
 1. A method for determining the nucleotide base sequence of aDNA molecule, comprising the steps of amplifying said DNA by apolymerase chain reaction, contacting the products of said amplifyingstep with an alkaline phosphatase, and determining the nucleotide basesequence of any polynucleic acid products of said amplifying step by achain termination procedure.
 2. The method of claim 1, wherein theproducts of said amplifying step are contacted with exonuclease I.